Elevated plasma free fatty acids (FFAs) resulting from obesity is a hallmark of the metabolic syndrome and contributes to type 2 diabetes. Data indicate that elevated plasma FFAs contribute to skeletal muscle pathologies associated with type 2 diabetes including inflammation, atrophy, and myopathy. Palmitate (PAL), the most abundant plasma FFA, serves as substrate for sphingolipid synthesis, and data indicate that elevation of plasma FFA as occurs in obesity and diabetes, increases muscle sphingolipids. Since sphingolipids comprise a diverse group of bioactive lipid mediators and impact cell programs including autophagy, apoptosis, inflammation, and proliferation, we hypothesize that PAL-mediated toxicity in skeletal muscle results in part via sphingolipid synthesis. Moreover, we propose that biological functions of distinct sphingolipids can be determined using specific pharmacological inhibition of pathways giving rise to these metabolites. To test the hypothesis we will 1) determine the impact of PAL on sphingolipids in a skeletal muscle model system and determine whether specific bioactive sphingolipids including ceramides and sphingosine-1-phosphate can be attenuated using pharmacological agents. We will then 2) determine gene expression changes induced by PAL and its sphingolipid metabolites, ceramide and sphingosine-1-phosphate, followed by identification of signaling pathways activated by these metabolites. Finally we propose 3) to use the diet-induced obesity mouse model, which demonstrates a time-dependent elevation of plasma fatty acids, to determine sphingolipid content in skeletal muscle of these animals, and determine whether they mirror the cell culture model in terms of sphingolipid profiles, activation of sphingolipid targets, and regulation of inflammatory markers and cytokines. We will then apply the same strategy in mice administered with the sphingolipid synthesis inhibitor myriocin, as well as in mice deficient for sphingosine kinase (SK1 -/- targeted gene deletion mice). These studies will provide novel insights into sphingolipid functions in skeletal muscle and will allow assessment of the feasibility of therapeutically targeting sphingolipid biosynthesis to preserve skeletal muscle health in the presence of elevated plasma FFA. Elevated plasma FFA contributes to myopathies, muscle wasting, muscle inflammation, and type 2 diabetes. Veterans suffer from type 2 diabetes at a higher rate than the general population. Therefore these studies are highly relevant to the VA mission. PUBLIC HEALTH RELEVANCE: Project Narrative Recent decades have witnessed a dramatic increase in obesity and metabolic disease (including metabolic syndrome, type 2 diabetes, atherosclerosis, cardiovascular disease). These health problems plague large sections of the population, and type 2 diabetes occurs at a higher rate in veterans than in the general United States population. A key feature of metabolic disease is the increase in plasma free fatty acids (FFA). This oversupply increases fatty acid deposition into peripheral tissues, including skeletal muscle. Normally, skeletal muscle serves a major function of plasma glucose uptake and utilization, and therefore, the health of this tissue plays a major role in diabetes. Moreover, in individuals who exhibit elevated plasma FFA, many muscle pathologies are observed, including fibrosis, wasting, inflammation, and myopathy. This proposal addresses the mechanism(s) by which FFA mediate these pathological processes in skeletal muscle. Specifically, we hypothesize that elevation of plasma FFA causes inappropriate synthesis of sphingolipids, key lipid mediators involved in numerous cell and organism processes. We propose to use cell culture and animal models to determine roles for specific sphingolipid signaling molecules in aberrant gene regulation and subsequent skeletal muscle pathology including inflammation and muscle wasting. This proposal directly addresses a primary health concern for veterans, namely, type 2 diabetes. In the general population, the incidence of diabetes is around 6%, (http://www.va.gov/pressrel/diabtsfs.htm). However, as found on the Department of Veterans Affairs Fact Sheet entitled "Advances in Diabetes Care", "Diabetes has particular importance for the Department of Veterans Affairs (VA) because the prevalence among VA patients -- one in six, or 16 percent -- is substantially higher than in the general population. . . VA is the largest integrated health care system to provide care to persons with diabetes." Therefore, there is little argument that diabetes and its sequelae represent a major concern for the VA. Furthermore, this disease is associated with aging, and as the average age of the population is increasing, DMII will become an even greater concern. Our proposal seeks to identify mechanisms of loss of skeletal muscle health in obesity, the metabolic syndrome, and diabetes. Completion of our specific aims will provide novel insights into the mechanisms by which these conditions decrease skeletal muscle health and function.